Cell migration assay

ABSTRACT

The present invention is directed to a modified cell migration assay allowing for improved identification and discrimination of chemokine receptor antagonists from non-specific blockers.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional applicationSerial No. 60/296,682 filed Jun. 7, 2001, which is incorporated hereinby reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention is directed to an assay for identifyingantagonists of chemoattractant receptors, such as chemokine receptors.One advantage of the assay compared with prior assays is its ability todiscriminate valid chemoattractant receptor antagonists from thosecompounds that generate false positive and negative signals.

BACKGROUND

[0003] High-throughput screening (HTS) methods for identifyingantagonists of chemoattractant receptors often rely on detectingperturbations in downstream events, such as cell migration. In the caseof chemokine receptors, leukocyte cell migration is often assayed.However, compounds disrupting cell membranes or blocking downstreamevents mimic these outcomes, masquerading as candidate antagonists.Considerable effort is then required to distinguish the genuineantagonists from those compounds or molecules that caused false positivesignals. Identifying true antagonists, which represent only a very smallfraction of the large collections of candidate antagonists analyzed inhigh-throughput screens, is a formidable task. Realizing any savings intime or expense can bring a new drug to patients more quickly and lessexpensively.

[0004] Conventional assays that are adapted for use in HTS methods forscreening small molecule antagonists of ligand-receptor interactions andsignaling are usually one-dimensional. That is, they isolate and assayonly the ligand-receptor interaction or the cellular signaling thatligand binding initiates, but not both. Because of this separation ofphysical interaction (ligand-receptor binding) from function (receptorsignaling and downstream events), false positive signals are oftenobserved, slowing discovery and development. False positives aremolecules that give the desired result for undesirable reasons; they areoften seen in screens for small molecule antagonists. Small moleculesthat initially appear to be inhibitors of receptor-ligand bindinginteractions (a desired result) may give such a result, for example,either by inhibiting the receptor-ligand interaction by binding thetarget receptor or ligand (desirable reasons), or by sickening orkilling cells, or wielding other undefined effects (undesirablereasons).

[0005] Furthermore, conventional drug discovery formats forchemoattractant receptor antagonists fail to identify all clinicallyimportant molecules, a consequence of false negative signals. Falsenegatives mean that clinically important molecules are undetected andremain undiscovered. For example, a molecule that permitschemoattractant receptor ligand-chemoattractant receptor binding, butinhibits chemoattractant receptor signaling, will be hidden in aninitial screen for inhibitors of ligand binding.

[0006] Chemoattractant molecules attract cells. For example, chemokines,a group of more than 40 small peptides (generally 7-10 kDa in size), actas molecular beacons for the recruitment, activation, and directedmigration of T lymphocytes, neutrophils and macrophages of the immunesystem, flagging pathogens and tumor masses for destruction. Whiledefending the individual from invading pathogens and tumors, the immunesystem can cause disease when improperly regulated. Chemokine-receptorbinding is linked to G-protein-coupled signaling cascades to mediatechemoattractant and chemostimulant signaling functions.

[0007] Inappropriate chemokine signaling can either promote infectionswhen not properly triggered (Forster et al., 1999) or lead to diseasesassociated with defective chemokine signaling, including asthma,allergic diseases, multiple sclerosis, rheumatoid arthritis, andatherosclerosis (reviewed in Rossi and Zlotnick, 2000). Becausechemokines play pivotal roles in inflammation and lymphocytedevelopment, the ability to specifically manipulate their activity willhave enormous impact on ameliorating and halting diseases that currentlyhave no satisfactory treatment. Chemokine receptor antagonists can beused to obviate the generalized and complicating effects of costlyimmunosuppressive pharmaceuticals in transplant rejection (reviewed inDeVries et al., 1999).

[0008] To expedite the identification of chemoattractant receptorantagonists, such as those for chemokine receptors, an assay that weedsout false signals by testing both chemoattractant receptor binding and abiological function would hasten drug development.

SUMMARY OF THE INVENTION

[0009] In a first aspect, the invention provides methods for identifyinga chemoattractant receptor antagonist. A cell having a chemoattractantreceptor is incubated with a candidate antagonist in the presence of anexcess of optimal ligand concentration for the chemoattractant receptor,and then cell migration is assayed. Cell migration indicates that thecandidate antagonist is an antagonist.

[0010] In one aspect, the invention provides methods for identifying achemokine receptor antagonist. A cell expressing a chemokine receptor isincubated with a candidate antagonist in the presence of an inhibitoryconcentration of chemokine ligand, and then cell migration is assayed.Cell migration indicates that the candidate antagonist is an antagonist.

[0011] The invention also provides for kits containing a solution withan inhibitory concentration for migration of chemokine for a chemokinereceptor bearing cell. In addition, such kits may also include a cellmigration apparatus.

[0012] In another aspect, the invention provides methods for identifyinga chemokine receptor antagonist. A candidate antagonist of a chemokinereceptor is first identified in a conventional assay. In a subsequentstep, the candidate antagonist is incubated with a chemokine receptorbearing cell in the presence of inhibitory concentration of ligand, andthen cell migration is assayed. Cell migration confirms that thecandidate antagonist is an antagonist.

[0013] These and other embodiments are discussed in detail below.

DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1. shows graphs depicting the selective activation of cellmigration by chemokine receptor antagonist by the (B)“reversed-activation of migration” (RAM) assay compared to (A)conventional assays.

[0015]FIG. 2. shows a graph depicting the dose response curve for CXCR4chemokine receptor-SDF ligand interaction, relating to cell migration.X-axis, chemokine concentration (expressed as log); Y-axis, cellmigration as measured in a cell migration assay (expressed as units offluorescence).

[0016]FIG. 3 shows a graph depicting representative curves thatdemonstrate the right-shift of the migration curve in the presence of anantagonist under RAM conditions. X-axis, chemokine concentration(expressed as log); Y-axis, cell migration as measured in a cellmigration assay (numbers of cells).

[0017]FIG. 4. depicts a schematic of a conventional cell migrationassay.

[0018]FIG. 5. shows graphs depicting the results from a RAM assayvalidation experiment using a protein CXCR4 antagonist. ChemokineSDF-mediated cell migration in the presence of the CXCR4 antagonist,vMIP-II, under (A) conventional and (B) RAM conditions.

[0019]FIG. 6. shows bar graphs depicting the results from a RAM assayvalidation experiment using small organic CXCR4 antagonists. ChemokineSDF-mediated cell migration in the presence of small organic moleculeCXCR4 antagonist (A) RAMAG-1, (B) RAMAG-2 and (C) RAMAG-3.

[0020]FIG. 7 demonstrates the efficacy of the RAM assay to discern falsepositive signals. (A) conventional assay, showing inactivation of cellmigration by three compounds known to be non-specific; (B) RAM assay,wherein the same three compounds are not indicative of a chemokinereceptor antagonist.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The reversed-activation of migration (RAM) assay of the inventionidentifies and discriminates antagonists while significantly decreasingthe prevalence of confounding false positive and negative signals foundin other assays. The time and labor involved to confirm a potentialpharmaceutical compound is therefore greatly reduced.

[0022] The methods of the invention include:

[0023] (1) incubating a cell comprising a chemoattractant receptor, suchas a chemokine receptor, with a candidate antagonist;

[0024] (2) contacting the cell with an inhibitory concentration of aligand for the chemoattractant receptor; and

[0025] (3) assaying cell migration.

[0026] Cell migration is used to identify the candidate antagonist as anantagonist.

[0027] The method may further comprise a “pre-step” in which theconcentration of a chemoattractant ligand (such as a chemokine) thatinhibits cell migration is determined, the “inhibitory concentration” ofa ligand for a chemoattractant receptor. Additional steps may be added,depending on the type of cell or agent being used, the assay, etc.

[0028] While conventional screens for antagonists of cell migrationmeasure the reduction of cell migration—a reduction in activity—RAMassays measure the activation of cell migration, an increase in activity(FIG. 1A, conventional migration assay; FIG. 1B, RAM assay). In the RAMassay, cells are challenged to migrate in the presence ofmigration-inhibitory concentrations of chemoattractants in response to acandidate antagonist; in a conventional assay, cells are challenged tomigrate in response to a chemoattractant in the presence of a candidateantagonist. A compound that gives a false positive signal in aconventional cell migration assay (inhibiting migration) will fail toactivate cell migration in the RAM format. In the RAM assay, only a trueantagonist activates migration. This distinction allows for simpleidentification of authentic antagonists.

[0029] Another advantage of the RAM assay is that the identifiedantagonists are more likely to be therapeutically useful than thoseidentified in conventional assays. A therapeutic chemoattractantreceptor antagonist is specific for that receptor, exerting its effectthrough the receptor. Such an antagonist reduces the effective affinitybetween the chemoattractant and the receptor without compromising thephysical integrity of the cell or completely disrupting the downstreamsignaling events leading to migration. A false positive identified in aconventional assay lacks at least one of these characteristics.

[0030] One possible explanation for the success of the RAM assay isbased on the observation that for a cell to migrate, the cell must havefront end-back end polarity. Such polarity is often initiated byextracellular signals, such as chemokines. For cell migration, thispolarity is achieved by a differential degree of chemoattractantreceptor occupancy at the two ends of the cell. However, highconcentrations of chemoattractant inhibit migration because allreceptors are occupied in all directions of the cell; the cell lacks adirectional cue. If increasing concentrations of ligand are plotted inrelation to cell migration, a bell-shape curve is observed (an exampleis shown in FIG. 2). A receptor antagonist that reduces the effectiveaffinity of a chemoattractant for a receptor allows the ligand to behavelike a ligand with lower affinity. The bell-shape curve, first observedin the absence of antagonists, shifts to the right in the presence ofincreasing concentrations of antagonist (see e.g., FIG. 3). This is onepossible explanation for the success of the present invention. Theinventors do not intend to be limited by this proposal.

Definitions

[0031] A “cell migration assay” tests the capacity of a cell to migratein response to a signal.

[0032] An “inhibitory concentration” of a chemoattractant is one thatinhibits cell migration. This concentration is greater than one thatactivates cell migration.

[0033] A “chemoattractant receptor” is a receptor that binds achemoattractant ligand, inducing cell migration. For example, achemokine receptor is a chemoattractant receptor whose chemoattractantligand is at least one chemokine.

[0034] In the following sections, the RAM assay is illustrated usingchemokines and chemokine receptors. However, any chemoattractant andchemoattractant receptor that induces cell migration may be used. TableA shows some examples of known chemoattractant receptors and some oftheir ligands. TABLE A Exemplary human chemoattractant receptors andexemplary ligands¹ Receptor Examples of ligands² BLT1 Leukotriene B4PDGFR Platelet-Derived Growth Factor FPR fMLP FPRL1 Unknown FMLPreceptor-like receptor Unknown CRTH2 prostaglandin D2 C3aR C3a C5aR C5aNoci-R Nociceptin EDG family Sphingosine 1-phosphate CB1 CannabinoidsVEGFR Vascular endothelial growth factor EGFR Epidermal growth factorFGFR Fibroblast growth factor P2Y receptor P2Y CTR Calcitonin CRLRCalcitonin gene-related peptide (CGRP) Histamine receptor HistamineThrombin receptor Thrombin TrkB Brain-derived neurotrophic factor (BDNF)

RAM Assay

[0035] In the RAM assay, a chemokine-bearing cell is incubated with acandidate antagonist and then contacted with an inhibitory concentrationof a ligand for the target chemokine receptor. The ability of the cellto migrate is then assayed. If the cell migrates in the presence of acandidate antagonist in the RAM assay, then a positive signal has beenobserved. “Antagonist” includes any molecule that partially or fullyblocks, inhibits, or neutralizes a biological activity, such as cellmigration. Similarly, “agonist” includes any molecule that mimics abiological activity of molecule, such as a chemokine. Molecules that canact as agonists or antagonists include small organic molecules,macromolecules, antibodies or antibody fragments, fragments or variantsof chemokines, peptides, etc. A “candidate antagonist” is a compoundthat is being tested for antagonist activity; likewise, a “candidateagonist” is a compound that is being tested for agonist activity.

[0036] Any cell migration assay format may be used, such as the ChemoTx®system (NeuroProbe, Rockville, Md.) or any other suitable device orsystem (Bacon et al., 1988; Penfold et al., 1999). In brief, these cellmigration assays work as follows. After harvesting and preparing thecells bearing the active target chemokine receptor, the cells are mixedwith candidate antagonists. The mixture is placed into the upper chamberof the cell migration apparatus. To the lower chamber, an inhibitoryconcentration of chemokine ligand is added. The migration assay is thenexecuted, terminated, and cell migration assessed.

[0037] To start the RAM assay, the solution of the inhibitoryconcentration of chemokine ligand is added to the lower chamber (6, FIG.4) of a cell migration apparatus, and the cell suspension is placed intothe upper chamber (4, FIG. 4) that is separated by a porous membrane (5,FIG. 4). The cells are incubated under culture conditions (37° C. forhuman cells) for 60 to 180 minutes in a humidified tissue cultureincubator. The incubation period depends on the cell type and ifnecessary, can be determined empirically.

[0038] At the end of the incubation period, the assay is terminated. Forexample, non-migrating cells on the upper chamber of the apparatus areremoved, using a rubber scraper or other manual method; enzymatically orchemically, e.g., EDTA and EGTA solutions. The membrane (5, FIG. 4) thatseparates the two chambers is then removed from the apparatus and rinsedwith Dulbecco's phosphate buffered saline (DPBS) or water. The number ofcells that migrate into the lower chamber is then determined.

[0039] The concentration of candidate antagonist to be screened in RAMassays may range from sub-nanomolar to millimolar. Screening acollection of small molecule compounds (such as a library synthesized bycombinatorial chemistry), the concentration of candidate antagonists istypically about 1-20 μM. “Compound” includes small inorganic and organicmolecules, macromolecules, peptides, proteins, polypeptides, nucleicacids, and antibodies.

Determining Inhibitory Concentrations of Ligand

[0040] A dose response of cell migration to a chemokine ligand can beperformed to define the inhibitory concentrations of a chemokine ligand.Any standard method for determining dose response curves can be used.One such method includes harvesting cells expressing the targetchemokine receptor, adding the cells to a cell migration device in thepresence of increasing amounts of chemokine, measuring cell migration,plotting cell migration versus chemokine concentration, and thencalculating from the graph those chemokine concentrations that inhibitcell migration.

[0041] As an example, a conventional cell migration assay, such as theChemoTx® system (NeuroProbe, Rockville, Md.) or any other suitabledevice or system (Bacon et al., 1988; Penfold et al., 1999) may be used.To obtain a dose response curve, cells expressing the target receptorare gathered. A chemokine ligand is prepared in a concentration seriesby serial dilution in a buffer. The concentration range is typicallybetween 0.1 nM and 10 mM, but will vary with ligand.

[0042] To start the cell migration assay, solutions of the variouschemokine ligand concentrations are added to the lower chamber (6, FIG.4) of a cell migration apparatus, and the cell suspension is placed intothe upper chamber (4, FIG. 4) that is separated by a porous membrane (5,FIG. 4). The cells are incubated under culture conditions (37° C. forhuman cells) for 60 to 180 minutes in a humidified tissue cultureincubator. The incubation period depends on the cell type and ifnecessary, can be determined empirically.

[0043] After terminating cell migration, non-migrating cells on theupper chamber of the apparatus are removed, using a rubber scraper orother manual method; enzymatically or chemically, e.g., EDTA and EGTAsolutions. The membrane (5, FIG. 4) that separates the two chambers isthen removed from the apparatus and rinsed with Dulbecco's phosphatebuffered saline (DPBS) or water. The number of cells that migrate intothe lower chamber is then determined.

[0044] Cell migration (Y-axis) is then plotted against the log(chemokineconcentration) (X-axis); a bell-shaped curve is observed (FIG. 2; seeExamples). From this plot (FIG. 2), the lowest concentration ofchemokine that inhibits cell migration can be determined. For ease ofreference, a second Y-axis (y₂, FIG. 2) can be drawn through the bellcurve, intersecting at its apex (maximal cell migration) and thecorresponding value on the X-axis. Those concentrations to the left ofthe Y₂-axis (lower) are stimulatory (2, FIG. 2); those to the right(higher) are inhibitory (3, shaded region, FIG. 2). These concentrationsare the “inhibitory concentrations” for cell migration (chemotaxis). Forexample, to determine the concentration at with migration is inhibitedby 90% of the maximum (to the right of the Y₂-axis, the “inhibitory”concentrations), the value corresponding to 10% of maximal cellmigration on the Y-axis is located. If the maximal cell migration signalis, e.g., 3.5×10⁴ cells, 10% thereof would be 350 (3.5×10⁴×0.1). Theinhibitory ligand concentration is then determined by locating thecorresponding X-axis coordinate. Preferably, the level of inhibition is50%, 60%, 70% or 80% of maximal cell migration. More preferably, thelevel of inhibition is 90% or even more preferably 95% or 100%inhibition as compared to the maximal signal for migration. Thedetermined chemokine concentration varies and depends on the nature ofthe receptor, the chemokine ligand and the target cell. Varying thedegree of chemotactic inhibition can be used to modulate the sensitivityof the RAM assay.

Application of RAM Assays in Comprehensive Screens for TherapeuticAntagonists

[0045] RAM assays can be performed in conjunction with any other assayused to screen for chemokine receptor antagonists. Not only is the RAMformat useful as a primary HTS step, but it also provides a confirmatoryor secondary assay for candidate antagonists identified in other assays.For example, a HTS method that measures Ca²⁺ mobilization, includingthose based on the FLIPR™ system (Molecular Devices Corp., Sunnyvale,Calif.) or other reporter-based methods which assay increases in freeintracellular Ca²⁺ levels, can be used as a primary assay. RAM assayscan be used to confirm such candidates, or vice-versa. As a secondaryassay, RAM would discriminate those candidate antagonists that exertnon-specific effects. When RAM assays are used with other HTS methods, ameans for discriminating true hits from non-specific blockers isprovided.

[0046] The RAM assay can be applied to any other assay format measuringcell migration or receptor activation, including methods that do notrequire migration of cells across a porous membrane. More usefultechnologies offering higher throughput and lower cost may be developedbased on use of the RAM concept.

Cells for Use in the RAM Assay

[0047] Cells expressing a target chemokine receptor (or chemoattractantreceptor) for use in the RAM assay may be gathered by a variety ofmethods, for example by centrifugation after collection from a subjector release from culture, and then resuspended in a buffer at anappropriate density, depending on cell type and cell size. Convenientcell concentrations range from about 1×10⁶ to 1×10⁷ cells/ml; oftenabout 2.5×10⁶ cells/ml is suitable.

Chemokine Receptors and Ligands

[0048] Cells that can be assayed in the RAM format include all thosethat express at least one chemokine receptor on the cell surface, suchas human monocytes, or other cells engineered to express recombinantchemokine receptors and are competent to activate cell migration. Knownchemokine receptors and some of their ligands are shown in Table B.Examples of chemokine receptors include, but are not limited to, the CXCclass, e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5; the CC class, CCR1,CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11; theCX3CR class, such as CX3CR1 and the XCR class, such as XCR1.

[0049] An example of a non-chemokine chemoattractant receptor is formylpeptide receptor like protein 1 (FPRL1); the ligands for which arew-peptide1 and w-peptide2 (Klein et al., 1998). Also see Table A forother examples. TABLE B Summary of the known chemokine receptors andsome of their known human ligands (Rossi and Ziotnik, 2000) ReceptorHuman ligands CXCR1 IL-8, GCP-2 CXCR2 IL-8, GCP-2, Gro α, Gro β, Gro γ,ENA-78, PBP CXCR3 MIG, IP-10, I-TAC CXCR4 SDF-1/PBSF CXCR5 BLC/BCA-1CCR1 MIP-1α, MIP-1β, RANTES, HCC-1, 2, 3, and 4 CCR2 MCP-1, MCP-2,MCP-3, MCP-4 CCR3 eotaxin-1, eotaxin-2, MCP-3 CCR4 TARC, MDC, MIP-1α,RANTES CCR5 MIP-1α, MIP-1β, RANTES CCR6 MIP-3α/LARC CCR7 MIP-3β/ELC,6Ckine/LC CCR8 I-309 CCR9 TECK XCR1 Lymphotactin CX3CR1Fractalkine/neurotactin CXCR6 CXCL16 CCR10 CTACK

[0050] Chemokines that can be used in the RAM assay include all knownchemokines. Examples of chemokines include, but are not limited to,IL-8, GCP-2, Gro α, Gro β, Gro γ, ENA-78, PBP, MIG, IP-10, I-TAC, SDF-1(PBSF), BLC (BCA-1), MIP-1α, MIP-1β, RANTES, HCC-1, -2, -3, and -4,MCP-1, -2, -3, and -4, eotaxin-1, eotaxin-2, TARC, MDC, MIP-3α (LARC),MIP-3β (ELC), 6Ckine (LC), I-309, TECK, lymphotactin, fractalkine(neurotactin), TCA-4, Exodus-2, Exodus-3 and CKβ-11.

[0051] Chemokine receptor/ligand combinations include those associatedwith inflammatory disorders, infectious diseases and transplantrejection. Such combinations include CX3CR1/fractalkine(transplantation), CCR5/MIP-1α, MIP-1β, or RANTES (HIV), CXCR4/SDF-1(HIV); and CCR7/MIP-3β, ELC or 6Ckine LC (inflammatory or allergicdiseases, e.g. asthma, multiple sclerosis, etc.).

Candidate Antagonists

[0052] Any molecule or compound can be screened for chemokine receptorantagonist activity. Compounds that inhibit chemokine receptor/ligandactivities, such as activating cell migration or modulatingintracellular Ca²⁺ concentrations are candidate antagonists.

[0053] Such molecules that may exert such antagonistic effects includesmall molecules that bind to chemokine receptors or their ligands.Examples of small molecule antagonists include small peptides,peptide-like molecules, preferably soluble and synthetic non-peptidylorganic or inorganic compounds. Other potential antagonist moleculesinclude nucleic acids such as aptamers and antibodies. These moleculesmay be collected into various libraries can be quickly screened fornovel chemokine receptor antagonists using the RAM assay.

[0054] Almost any antibody (Ab) that inhibits chemotactic cell migrationis also a candidate antagonist. Examples of antibody antagonists includepolyclonal, monoclonal, single-chain, anti-idiotypic, chimeric Abs, orhumanized versions of such Abs or fragments. Abs may be from any speciesin which an immune response can be raised. Humanized Abs areexceptionally well-adapted for treatment of diseases and representattractive candidate antagonists (Jones et al., 1986; Riechmann et al.,1988; Verhoeyen et al., 1988); (U.S. Pat. No. 481,6567, 1989). Suchantibodies may bind to chemokine receptors to inhibit cell migration.

[0055] Alternatively, a potential antagonist or agonist may be a closelyrelated protein, for example, a mutated form of a chemokine receptorligand or other protein that recognizes a chemokine receptor interactingprotein, but imparts no effect, thereby competitively inhibitingchemokine receptor action.

[0056] Aptamers are short oligonucleotide sequences that can be used torecognize and specifically bind almost any molecule, such molecules mayalso act antagonistically. The systematic evolution of ligands byexponential enrichment (SELEX) process (Ausubel et al., 1987; Ellingtonand Szostak, 1990; Tuerk and Gold, 1990) is powerful and can be used tofind such aptamers. Aptamers have many diagnostic and clinical uses,including as antagonists. In addition, they are inexpensive tomanufacture and can be easily applied in a variety of formats, includingadministration in pharmaceutical compositions, bioassays, and diagnostictests (Jayasena, 1999). The RAM assay can also be used as a screen toisolate aptamers de novo.

Quantifying Migratory Cells

[0057] Quantifying migratory cells may be accomplished by a largevariety of available methods, such as those that assay the amount ofDNA, (e.g., the CyQuant Cell Proliferation Kit (Molecular Probes)) andthen assaying the generated signal, such as fluorescence. Other methodsinclude counting the cells using a microscope, or labeling cells with asuitable detectable marker, such as dyes (such as Calcein AM(NeuroProbe) or the many labels available from Molecular Probes (Eugene,Oreg.)) or radioactive labeling (e.g. cell surface iodination with ¹³⁵I,protein synthesis labeling with ³⁵S-methionine/³⁵S-cysteine or nucleicacid labeling with ³II).

Buffers and Cell Culture Media

[0058] Buffers that may be used to prepare the various solutions includecell culture media, although serum or other growth and chemotacticfactors may be removed so that the results in a cell migration assay arenot confounded and can be mostly attributable to the chemokine-chemokinereceptor interaction. In some cases, a protein may be added to supportthe cells, such as various albumins, including bovine serum albumin.Optimal media selection depends on the cell type; that media used toculture the cells usually represents a preferred option. Examples ofsuitable culture media include Iscove's Modified Dulbecco's Medium(IMDM), Dulbecco's Modified Eagle's Medium (DMEM), Minimal EssentialMedium Eagle (MEM), Basal Medium Eagle (BME), Click's Medium, L-15Medium Leibovitz, McCoy's 5A Medium, Glasgow Minimum Essential Medium(GMEM), NCTC 109 Medium, Williams' Medium E, RPMI-1640, and Medium 199.A medium specifically developed for a particular cell type/line or cellfunction, e.g. Madin-Darby Bovine Kidney Growth Medium, Madin-DarbyBovine Kidney Maintenance Medium, various hybridoma media, EndothelialBasal Medium, Fibroblast Basal Medium, Keratinocyte Basal Medium, andMelanocyte Basal Medium are also useful. If desired, a protein-reducedor free and/or serum free medium and/or chemically defined, animalcomponent free medium may be used, e.g., CHO, Gene Therapy Medium orQBSF Serum-free Medium (Sigma Chemical Co.; St. Louis, Mo.), DMEMNutrient Mixture F-12 Ham, MCDB (105, 110, 131, 151, 153, 201 and 302),NCTC 135, Ultra DOMA PF or HL-1 (both from Biowhittaker; Walkersville,Md.), may be used.

[0059] If desired, the media may be further supplemented with reagentsthat limit acidosis of the cultures, such as buffer addition to themedium (such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid(BES), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris),N-(2-hydroxyethyl)piperazine-N′3-propanesulfonic acid (EPPS or HEPPS),glyclclycine, N-2-hydroxyehtylpiperazine-N′-2-ethanesulfonic acid(HEPES), 3-(N-morpholino)propane sulfonic acid (MOPS),piperazine-N,N′-bis(2-ethane-sulfonic acid) (PIPES), sodium bicarbonate,3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid)TAPSO, (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES),N-tris(hydroxymethyl)methyl-glycine (Tricine),tris(hydroxymethyl)-aminomethane (Tris), etc.). Frequent medium changesand changes in the supplied CO₂ (often approximately 5%) concentrationmay also be used to control acidosis.

Kits

[0060] Components to carry out RAM assays may be assembled into kits,containers, packs, or dispensers together with instructions foradministration. When supplied as a kit, the different components of thecomposition may be packaged in separate containers and admixedimmediately before use. Such packaging of the components separately maypermit long-term storage without losing the active components'functions. For example, a kit may include a cell migration apparatus, achemokine receptor-bearing cell and a solution comprising an inhibitorymigratory concentration of chemokine for the chemokine receptor bearingcell. The solution may be supplied lyophilized.

[0061] (a) Containers or Vessels

[0062] The reagents included in the kits can be supplied in containersof any sort such that the life of the different components arepreserved, and are not adsorbed or altered by the materials of thecontainer. For example, sealed glass ampules may contain lyophilizedchemokine or a buffer that has been packaged under a neutral,non-reacting gas, such as nitrogen. Ampules may consist of any suitablematerial, such as glass, organic polymers, such as polycarbonate,polystyrene, etc., ceramic, metal or any other material typicallyemployed to hold reagents. Other examples of suitable containers includesimple bottles that may be fabricated from similar substances asampules, and envelopes, that may consist of foil-lined interiors, suchas aluminum or an alloy. Other containers include test tubes, vials,flasks, bottles, syringes, or the like. Containers may have a sterileaccess port, such as a bottle having a stopper that can be pierced by ahypodermic injection needle. Other containers may have two compartmentsthat are separated by a readily removable membrane that upon removalpermits the components to mix; for example, lyophilized chemokine in onecompartment, and a buffer or water in the other. Removable membranes maybe glass, plastic, rubber, etc.

[0063] (b) Instructional Materials

[0064] Kits may also be supplied with instructional materials.Instructions may be printed on paper or other substrate, and/or may besupplied as an electronic-readable medium, such as a floppy disc,CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, etc. Detailedinstructions may not be physically associated with the kit; instead, auser may be directed to an internet web site specified by themanufacturer or distributor of the kit, or supplied as electronic mail.

EXAMPLES

[0065] The following examples are intended to illustrate and validatethe RAM assay concept of the present invention without limitation. Thechemoattractant receptor and ligands used to illustrate the inventionare chemokine receptors and chemokines. However, any chemoattractantligand for any chemoattractant receptor may be used. For examples, seeTable A.

[0066] Examples 1, 2 and 4 demonstrate the effectiveness of the RAMassay, testing specific and non-specific antagonists of CXCR4 asdiscovered in conventional assays. Example 3 demonstrates the broadapplicability of chemoattractant receptors by examining three chemokinereceptors.

EXAMPLE 1 Determining Inhibitory Concentration of SDF (CXCR4)

[0067] To obtain a dose response curve for activated lymphocytesexpressing cell surface CXCR4, a conventional cell migration assay wasused (Bacon et al., 1988; Penfold et al., 1999). Cells were harvested bycentrifugation and then resuspended in cell migration buffer (Hank'sbalanced salt solution (HBSS)/0.1% bovine serum albumin (BSA) at 2.5×10⁶cells/ml. The CXCR4 ligand stromal-derived factor (SDF-1) was preparedin a concentration series (0.1 nM to 10 mM) by serial dilution in cellmigration buffer. At low concentrations, SDF activates cell migration ofCXCR4-bearing activated lymphocytes.

[0068] SDF ligand was loaded in the bottom chamber of a ChemoTx® cellmigration apparatus (5 μm pore polycarbonate polyvinylpyrrolidone-coatedfilters (Neuroprobe; Gaithersburg, Md.); 29 μl/well) and 20 μl of cellsuspension was placed in the upper chamber. The cells were incubated at37° C. for 150 minutes. The assay was terminated by removing the cellsfrom the upper chamber and membrane surface using a rubber scraper. Thecells that migrated to the lower chamber were quantified by the CyQuantassay (Molecular Probes; Eugene, Oreg.), a fluorescent dye method thatmeasures nucleic acid content.

[0069] To determine the minimum concentration of SDF to inhibit cellmigration, chemokine concentration (X-axis) is plotted against relativefluorescent units (RFUs), correlating to the number of cells migrating(Y-axis) (FIG. 2). Initially as SDF concentration increases, cellmigration increases linearly (2, FIG. 2); however, at higherconcentrations (3, FIG. 2), migration levels first flatten and thendecrease until migration is barely detectable. This bell-shaped curve istypical of chemokine and chemokine receptor-mediated cell migration. Inthis experiment, 1 μM of SDF was determined to be completely inhibitory;the inhibitory concentration range was 200 nM to 1 μM.

EXAMPLE 2 Validation of the RAM Assay Using a Viral PolypeptideAntagonist of CXCR4

[0070] In the RAM assay, antagonists of chemokine receptors areidentified by their ability to activate migration of cells that areincubated with inhibitory chemokine concentrations. To validate the RAMassay, the viral chemokine, vMIP-II, was used as a CXCR4 antagonist.vMIP-II binds with high affinity to CXCR4, blocking receptor signalingand inhibiting cell migration, competing with CXCR4's usual ligand, SDF(Kledal et al., 1997). If CXCR4 expressing cells that are immobilized byinhibitory concentrations of SDF are activated to migrate in thepresence of vMIP-II with increased migration, this result would verifythe RAM assay principle. For reference and as a control, a conventionalcell migration assay was performed. In the conventional assay format,cell migration is inhibited by vMIP-II.

[0071] Cell migration was measured using the two formats with thecorresponding amounts of SDF chemokine:

[0072] (1) a conventional assay (control); 1 nM SDF; and

[0073] (2) a RAM assay, 1 μM SDF.

[0074] Activated lymphocytes expressing cell surface CXCR4 wereharvested as in Example 1. For the conventional assay, a concentrationseries of vMIP-II was first mixed with activated lymphocytes, and thesolution then placed in the upper chamber of a ChemoTx® cell migrationapparatus (5 μm pore polycarbonate polyvinylpyrrolidone-coated filters(Neuroprobe), 20 μl/well); 29 μl of a 1 nM solution of SDF was placed inthe lower chamber. For the RAM assay, the cells were prepared as for theconventional assay, except the SDF concentration in the lower chamberwas 1 μM. The cells were incubated at 37° C. for 150 minutes. The assaywas terminated by removing the cells from the upper chamber and membranesurface using a rubber scraper. The cells that migrated to the lowerchamber were quantified by the CyQuant assay (Molecular Probes).

[0075] In the conventional assay (FIG. 5A), cell migration was partiallyinhibited at 11 μM of vMIP-II; cell migration was further inhibited asthe vMIP-II concentration increased (up to 100 nM), verifying thatvMIP-II is an antagonist of CXCR4. In the RAM assay format (FIG. 5B),little migration was observed in the absence of vMIP-II. However,migration was activated in the presence of vMIP-II at 11 nM, mirroringthe decrease of migration seen in the conventional assay (FIG. 5A).Increased vMIP-II concentration correlated with an increase in cellmigration, with maximal migration being observed at 100 nM.

EXAMPLE 3 Validation of RAM Assay Using Known Small Molecule Antagonistsof CXCR3, CXCR4 and CCR1

[0076] RAM assays were performed as described in Example 2, exceptpreviously identified small molecule antagonists instead of vMIP-II wereused, as well as additional cell types as described in Table 2. TABLE 2Experimental variables Antagonist¹ Receptor Ligand(s) Cells RAMAG-1CXCR3 I-TAC (250 activated human lymphocytes nM), IP-10 RAMAG-2 CXCR4SDF-1 CXCR4-expressing MOLT-4 cells (human T lymphoblast; American TypeTissue Collec- tion (ATCC); Manassas, VA) RAMAG-3 CCR1 MIP-1α THP-1cells (human monocytic; ATCC)

[0077] In the RAM assay, activated lymphocytes incubated in the presenceof increasing concentrations of RAMAG-1 and the CXCR3 ligand I-TAC at250 nM, cell migration was activated at less than 1 μM (FIG. 6A); asRAMAG-1 concentration increased, migration increased, reachingapproaching a maximum at (−6.5 to −5) of RAMAG-1.

[0078] At a CXCR4 SDF-1 ligand concentration of 100 nM usingCXCR4-expressing MOLT-4 cells, RAMAG-2 activated cell migration at 5 μM(FIG. 5, B). As was observed with RAMAG-1, further activation ofmigration was seen as the RAMAG-2 concentration increased to 10 μM.

[0079] The CCR1 antagonist, RAMAG-3 also gave similar results. In a RAMassay using CCR1-expressing THP-1 cells, RAMAG-3 activated cellmigration at 100 nM; as RAMAG-3 concentration increased, so did themigration signal (FIG. 5, C).

EXAMPLE 4 Validation of RAM Assay Using Known Small Molecules thatNon-specifically Inhibit Cell Migration in CXCR4-bearing Cells inConventional Assays

[0080] This experiment conclusively demonstrates the ability of the RAMassay to discern non-specific and specific chemokine receptorantagonists. A conventional and RAM assays were performed as describedin Example 2, but with the following candidate antagonists:

[0081] (1) control (no candidate antagonist)

[0082] (2) positive control (vMIP-II; a known CXCR4 antagonist)

[0083] (3) Known non-specific inhibitors of cell migration:

[0084] compound #1

[0085] compound #2

[0086] compound #3.

[0087] As shown in FIG. 7A, control cells migrated, but those incubatedwith vMIP-II and compounds #1, #2 and #3 showed decreased cellmigration. When these same candidate antagonists were subjected to a RAMassay (FIG. 7B), control cells did not migrate, as expected, whilevMIP-II-treated cells did migrate (also expected). However, compounds#1, #2 and #3, known compounds that non-specifically inhibit cellmigration in conventional assays, failed to activate cell migration inthe RAM assay.

[0088] From the results presented in Examples 2-4, the RAM assaydistinguishes between non-specific and specific antagonists ofchemoattractant receptors, such as chemokine receptors.

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1. A method for identifying a chemoattractant receptor antagonist,comprising: incubating a cell comprising a chemoattractant receptor witha candidate antagonist; contacting the cell with an inhibitoryconcentration of a ligand for the chemoattractant receptor; and assayingcell migration, wherein cell migration identifies the candidateantagonist as an antagonist.
 2. The method of claim 1, wherein thecandidate antagonist is a small peptide, peptide-like molecule,non-peptidyl organic compound, inorganic compound, nucleic acid orantibody.
 3. The method of claim 1, wherein the inhibitory concentrationof a ligand for the chemoattractant receptor inhibits cell migrationgreater than or equal to 50% of maximal ligand-activated cell migration.4. The method of claim 1, wherein the inhibitory concentration of aligand for the chemoattractant receptor inhibits cell migration greaterthan or equal to 95% of maximal ligand-activated cell migration.
 5. Themethod of claim 1, wherein the inhibitory concentration of a ligand forthe chemoattractant receptor inhibits cell migration is 100% of maximalligand-activated cell migration.
 6. A method for identifying a chemokinereceptor antagonist, comprising: incubating a cell comprising achemokine receptor with a candidate antagonist; contacting the c ellwith an inhibitory concentration of a ligand for the chemokine receptor;and assaying cell migration, wherein cell migration identifies thecandidate antagonist as an antagonist.
 7. The method of claim 6, whereinthe candidate antagonist is a small peptide, peptide-like molecule,non-peptidyl organic compound, inorganic compound, nucleic acid orantibody.
 8. The method of claim 6, wherein the chemokine receptor isselected from the group consisting of CCR, CXCR, CX3CR or XCR classes ofchemokine receptors.
 9. The method of claim 8, wherein the chemokinereceptor is CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR1, CCR2, CCR3, CCR4,CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1 or XCR1.
 10. Themethod of claim 6, wherein the chemokine is selected from the groupconsisting of CCR, CXCR, and CX3CR ligands.
 11. The method of claim 10,wherein the chemokine is IL-8, GCP-2, Gro α, Gro β, Gro γ, ENA-78, PBP,MIG, IP-10, I-TAC, SDF-1, BLC, MIP-1α, MIP-1β, RANTES, HCC-1, HCC-2,HCC-3, HCC-4, MCP-1, MCP-2, MCP-3, MCP-4, eotaxin-1, eotaxin-2, TARC,MDC, MIP-3 α, MIP-3β, 6Ckine, I-309, TECK, lymphotactin, fractalkine,TCA-4, Exodus-2, Exodus-3 or CKβ-11.
 12. The method of claim 6, whereinthe chemokine receptor comprises CCR5, and the chemokine comprisesMIP-1α, MIP-1β, or RANTES.
 13. The method of claim 6, wherein thechemokine receptor comprises CXCR4, and the chemokine comprises SDF-1.14. The method of claim 6, wherein the chemokine receptor comprisesCCR7, and the chemokine comprises MIP-3β, ELC or 6Ckine.
 15. The methodof claim 6, wherein the inhibitory concentration of a ligand for thechemokine receptor inhibits cell migration greater than or equal to 50%of maximal ligand-activated cell migration.
 16. The method of claim 6,wherein the inhibitory concentration of a ligand for the chemokinereceptor inhibits cell migration greater than or equal to 95% of maximalligand-activated cell migration.
 17. The method of claim 6, wherein theinhibitory concentration of a ligand for the chemokine receptor inhibitscell migration is 100% of maximal ligand-activated cell migration.
 18. Akit, comprising: a cell migration apparatus and a solution comprising aninhibitory concentration of chemokine for a chemokine receptor bearingcell.
 19. The kit of claim 18, wherein the solution is lyophilized. 20.A method for identifying a chemokine receptor antagonist, comprising:identifying a candidate antagonist of a chemokine receptor in aconventional assay; further comprising a second step comprising:incubating a cell comprising the chemokine receptor with the candidateantagonist; contacting the cell with an inhibitory concentration of aligand for the chemokine receptor; and assaying cell migration, whereincell migration identifies the candidate antagonist as an antagonist.